Detection system based on CRISPR-Cas10 compound and graphene field effect transistor and detection method thereof

Abstract

The invention relates to a detection system based on a CRISPR-Cas10 compound and a graphene field effect transistor and a detection method thereof. The detection system comprises a CRISPR-Cas10 compound and a CRISPR-GFET biosensor, the CRISPR-GFET biosensor comprises a sensing substrate, a graphene transistor sensing chip is arranged on the sensing substrate, and a micro-fluidic chip is arranged on the graphene transistor sensing chip; a CRISPR system mixing hole and a detection reaction hole which are connected through a flow channel are formed in the micro-fluidic chip, and a source electrode and a drain electrode are arranged on the two sides of the detection reaction hole respectively; a gold nano material and a hairpin reporter are fixed in the detection reaction hole, and a gate electrode is arranged in the detection reaction hole. According to the detection system, high integration of the detection process is realized, and the experimental operation is remarkably simplified. The sample transfer and external operation steps are reduced, the pollution risk is reduced, the detection efficiency and stability are improved, and the application potential of the detection chip in instant detection is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a detection system and method based on a CRISPR-Cas10 complex and a graphene field-effect transistor. Background Technology

[0002] Clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems have sparked considerable interest in developing CRISPR-based molecular diagnostics due to their superior nucleic acid targeting capabilities. Unlike Cas9, a trans-cleavage mechanism has been discovered in type V (Cas12a) and type VI (Cas13a) CRISPR systems, which can indiscriminately cleave any available non-target single-stranded DNA (ssDNA; Cas12a) or RNA (ssRNA; Cas13a). Although this process operates in a multi-flip mode, rapidly generating sensor signals, it is accompanied by the cleavage of the target nucleic acid molecular backbone, leading to the inactivation of Cas12a and Cas13a and hindering continued trans-cleavage. The unique feature of type III CRISPR systems is their target RNA-dependent mechanism, which protects the host from nucleic acid invasion and exhibits good stability when stored at room temperature. More importantly, similar to type VI systems, they specifically recognize RNA while activating the HD nuclease domain within the Cas10 subunit, mediating trans-cleavage of DNA, which has recently attracted wider attention in the field of nucleic acid detection.

[0003] Currently, most CRISPR-based nucleic acid detection platforms rely on pre-amplification processes of the target nucleic acid, such as polymerase chain reaction (PCR) or isothermal amplification methods (e.g., recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP)). These methods typically involve cumbersome procedures, stringent experimental conditions, and sophisticated equipment, resulting in long turnaround times, high operating costs, and an increased risk of sample contamination. To overcome these limitations, recent research has combined CRISPR systems with electrochemical or optical sensors, demonstrating a variety of CRISPR-based amplification-free biosensors. However, these electrochemical or optical biosensors often require complex device designs, multi-step reactions, or appropriate indicator molecules to convert lysis events into measurable signals. This hinders their widespread application, and the bulky and expensive optical equipment also limits their ability to facilitate point-of-care diagnostics and field analysis.

[0004] Field-effect transistors (FETs) possess advantages such as rich analyzable signals, low cost, low power consumption, small size, and good compatibility with integrated circuits, promising to provide more opportunities for biomolecular detection. In recent years, graphene-based field-effect transistor (G-FET) biosensors have attracted considerable attention for their rapid analysis of nucleic acid information and high-precision detection of various diseases, thanks to the advantages of graphene's ultrathin sensing layer, high charge carrier mobility, and good biocompatibility. However, traditional GFET-based nucleic acid detection biosensors rarely achieve 10-1 ppm in large amounts of buffer solution or diluted biological fluids. -16 Limit of detection (LOD) for M.

[0005] Therefore, there is an urgent need to develop a simple, rapid, and sensitive nucleic acid detection platform to overcome the limitations of existing strategies. Researching an amplification-free nucleic acid detection technology platform and detection method based on the synergy of CRISPR-Cas10 and graphene field-effect transistors is of great significance for nucleic acid detection. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a detection system and method based on a CRISPR-Cas10 composite and a graphene field-effect transistor.

[0007] The technical solution of the present invention is as follows:

[0008] A detection system based on a CRISPR-Cas10 complex and a graphene field-effect transistor, comprising a CRISPR-Cas10 complex and a CRISPR-GFET biosensor.

[0009] The CRISPR-GFET biosensor includes a sensing substrate, on which a graphene transistor sensing chip is disposed, and on which a microfluidic chip is disposed; the microfluidic chip is provided with a CRISPR system mixing hole and a detection reaction hole connected by a flow channel, and a source electrode and a drain electrode are respectively disposed on both sides of the detection reaction hole.

[0010] The detection reaction well contains gold nanomaterials and a hairpin reporter, and is also equipped with a gate electrode.

[0011] According to a preferred embodiment of the present invention, the CRISPR-GFET biosensor has a size of 30mm × 12mm; the CRISPR system mixing orifice and detection reaction orifice have a size of 1.5mm × 1.5mm; and the flow channel has a size of 0.5mm × 3mm.

[0012] According to a preferred embodiment of the present invention, the gate electrode is a silver / silver chloride reference electrode or a metal electrode, which is placed in the detection reaction well.

[0013] According to a preferred embodiment of the present invention, the gold nanomaterials (AuNPs) are prepared according to the following method:

[0014] Prepare a 38.8 mM sodium citrate aqueous solution and a 1 mM HAuCl4 aqueous solution. Heat the HAuCl4 aqueous solution to boiling, and then add the sodium citrate aqueous solution to the HAuCl4 aqueous solution while stirring. React for 10 min while heating to boiling. After the reaction is complete, stir for another 15 min, cool to 25 °C, and filter to obtain gold nanomaterials (AuNPs).

[0015] According to a preferred embodiment of the present invention, the hairpin reporter is a TCEP-pretreated thiolized hairpin DNA reporter gene, prepared as follows: First, the hairpin reporter is dissolved in annealing buffer (10 mM Tris, pH 7.5-8.0, 50 mM NaCl, 1 mM EDTA) to prepare a 15 μM hairpin DNA reporter gene solution, heated at 95 °C for 2 min, and then slowly cooled to 25 °C to form a secondary structure, obtaining a thiol-modified hairpin reporter gene; finally, 200 μl of the 15 μM thiol-modified hairpin reporter gene is mixed with 5 μl of 1 M TCEP solution, and reduced at 25 °C for 30 min to obtain a TCEP-pretreated thiolized hairpin DNA reporter gene;

[0016] The sequence of the hairpin DNA reporter gene is as follows:

[0017] 5'-TCAGCGTTTCCTTTACCATTTTTTTAACTTATTTGGGTTTTTTTTTT-3'.

[0018] According to a preferred embodiment of the present invention, the preparation method of the CRISPR-Cas10 complex includes the following steps:

[0019] (1) RNA-F / R / SR primers were used for overlap extension PCR amplification to obtain an artificial mini CRISPR fragment; the linearized pUCE plasmid was used as a template and pUCE-repeat-F / R primers were used for PCR amplification to obtain a pUCE fragment with Ld repeat sequences at both ends.

[0020] (2) The artificial mini CRISPR fragment was ligated to the pUCE fragment containing Ld repeat sequences at both ends by Gibson assembly to obtain the pUCE-S-RNA plasmid;

[0021] (3) pUCE-S-RNA plasmid, pET30a-Csm2 plasmid and p15AIE-Cas-Csm3 plasmid D34AThe plasmid was transformed into Escherichia coli BL21(DE3) to obtain an engineered bacterium expressing the CRISPR-Cas10 complex;

[0022] (4) The engineered bacteria expressing the CRISPR-Cas10 complex were activated and cultured for 12-16 h to obtain the culture. The culture was then transferred to TB liquid medium and cultured until OD600 = 0.6-0.8. IPTG was added and cultured for another 16-20 h. After centrifugation, column chromatography and elution, the CRISPR-Cas10 complex was obtained.

[0023] More preferably, in step 1), the sequence of the RNA-F / R / SR is:

[0024] RNA-F:

[0025] 5'-GCTAACAACTTATCTCCGCTAGAAGGAGACGAGAACGCTAGTTACACTA-3',

[0026] RNA-R:

[0027] 5'-GTTCTCGTCTCCTTCTAGCGGAGATAAGTTGTTAGCACACAATCGAAGC-3';

[0028] RNA-SR:

[0029] 5'-TAGCACACAATCGAAGCGCAGTAAGGATGGCTAGTGTAACTAGCG-3';

[0030] The pUCE-repeat-F / R sequence is as follows:

[0031] pUCE-repeat-F:

[0032] 5'-GCTAGAAGGAGACGAGAACAAGCTTGCGGCCGCACTC-3',

[0033] pUCE-repeat-R:

[0034] 5'-GTTCTCGTCTCCTTCTAGCGGAGATAAGTTGTTAGCTTAATGCTAA-3'.

[0035] According to a preferred embodiment of the present invention, the method for fabricating the CRISPR-GFET biosensor includes the following steps:

[0036] 1) Two parallel, highly conductive laser-induced graphene (LIG) films were induced on polyimide (PI) using a laser to obtain a LIG / PI film;

[0037] 2) Polydimethylsiloxane (PDMS) monomer and curing agent are deposited on a silicon mold and cured at 75-85°C for 55-65 min to obtain cured PDMS; the cured PDMS is laser-engraved and cut, and interconnected CRISPR system mixing holes and detection reaction holes are prepared in the middle to obtain a PDMS electrode adhesion layer; the PDMS electrode adhesion layer is pasted on a LIG / PI film, and then the PI film is peeled off to obtain a LIG / PDMS layer; the LIG / PDMS layer is then bonded to a graphene substrate to obtain a GFET chip;

[0038] 3) Gold nanomaterials (AuNPs) are deposited in the detection reaction wells of the GFET chip to obtain an AuNPs-modified GFET chip; then the AuNPs-modified GFET chip is co-incubated with hairpin reporter (re-hpDNA) for 12-16 hours to obtain a re-hpDNA-modified chip; the re-hpDNA-modified chip is then chemically blocked to obtain a CRISPR-GFET biosensor.

[0039] More preferably, in step 1), the mass ratio of polydimethylsiloxane to curing agent is 5:1.

[0040] More preferably, in step 2), the laser power for laser engraving and cutting is 25–35 W, and the scanning speed is 25–30 mm / s. -1 .

[0041] More preferably, in step 3), the concentration of the hairpin reporter is 1–10 μM.

[0042] More preferably, in step 3), the blocking is: first, the chip is modified with 2mM MCH and re-hpDNA and then incubated for 1 to 1.5 hours, and then modified with 1% BSA and re-hpDNA and incubated for 30 to 60 minutes.

[0043] The above-mentioned detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor is applied to the detection of RNA or miRNA.

[0044] According to a preferred embodiment of the present invention, the miRNA is miRNA-155;

[0045] The sequence of miRNA-155 is: 5'-UUAAUGCUAAUCGUGAUAGGGGUU-3'.

[0046] A method for detecting RNA or miRNA using the above-mentioned detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor includes the following steps:

[0047] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0048] b. Add the sample to be tested, the CRISPR-Cas10 complex, and the lysis buffer to DEPC water and mix thoroughly to obtain the CRISPR reaction system. Then, add the CRISPR reaction system to the CRISPR system mixing well of the CRISPR-GFET biosensor and mix for 5–10 min. Then, blow the mixed sample into the detection reaction well and incubate at 37°C for 15–75 min. After cleaning the detection reaction well, measure the transfer characteristic curve of the CRISPR-GFET biosensor to obtain the Dirac point value D2.

[0049] c. Calculate ΔD = D2 - D1 and record the resulting difference, the Dirac point voltage difference ΔD, as the sensor output signal; when ΔD is positive, the target RNA is present in the sample to be detected; when ΔD is negative or equal to 0, the target RNA is not present in the sample to be detected.

[0050] According to a preferred embodiment of the present invention, in step b, the lysis buffer comprises: 50–100 nM Tris-HCl (pH = 7), 10–20 mM MgCl2, 50–100 mM KCl, and 1–2 mg / ml BSA.

[0051] According to a preferred embodiment of the present invention, in step b, the volume ratio of the sample to be tested, the CRISPR-Cas10 complex, the lysis buffer, and the DEPC water is (1-2):(1-2):(1-2):(4-7); the final concentration of the CRISPR-Cas10 complex in the CRISPR reaction system is 10-25 nM; and the dilution factor of the sample to be tested is 0.1-0.2.

[0052] Where this invention is not detailed, existing technologies may be used.

[0053] The technical features of this invention are as follows:

[0054] like Figure 1As shown, the crRNA in the CRISPR-Cas10 complex provided by this invention can specifically recognize the target RNA. Therefore, when the target RNA is present, it binds to the crRNA in the CRISPR-Cas10 complex, activating the CRISPR-Cas10 complex. This activation process causes the CRISPR-Cas10 complex to cleave hairpin reporters fixed on the graphene surface. As a large number of hairpin reporters cleave away from the graphene surface, the electron doping of the hairpin reporters on the graphene surface decreases sharply, leading to a downward shift of the Fermi level in the graphene, and ultimately causing a positive shift of the Dirac point.

[0055] Conversely, non-target RNAs, lacking pre-designed CRISPR target sites, cannot activate the CRISPR-Cas10 complex to cleave the immobilized hairpin reporter, resulting in no movement of the Dirac point.

[0056] The beneficial effects of this invention are as follows:

[0057] 1. This invention provides a novel detection system based on a CRISPR-Cas10 composite and a graphene field-effect transistor, which achieves a high degree of integration in the detection process and significantly simplifies experimental operations. It not only reduces sample transfer and external operation steps, lowering the risk of contamination, but also improves detection efficiency and stability, enhancing the application potential of the detection chip in point-of-care testing (POCT).

[0058] 2. The CRISPR-GFET biosensor fabrication process provided by this invention is simple, avoiding complex operations such as photolithography and vapor deposition. Device fabrication can be achieved in a single bonding step. Furthermore, it has low fabrication costs and minimal environmental pollution, avoiding the use of graphene oxide precursors and metal electrodes, and eliminating the need for localized treatments and patterning chemicals, thus reducing environmental pollution. Moreover, due to the programmability of the CRISPR system, the CRISPR-GFET biosensor is versatile and suitable for large-scale industrial production.

[0059] 3. The detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor provided by this invention effectively prevents the degradation of the RNA sample to be tested by using the CRISPR-Cas10 complex, maintains the activity of the Cas10 polymerase to continuously cleave the DNA reporter, avoids the decrease in detection sensitivity due to the decrease in the concentration of the RNA sample to be tested, and also utilizes hairpin DNA with high charge density as a reporter, thereby enhancing the detection signal. It can effectively distinguish between healthy people and breast cancer patients, and eliminates the need for extraction, purification and amplification processes, thereby shortening the detection time and avoiding the risks of nucleic acid amplification and cross-contamination, making it suitable for clinical use.

[0060] 4. The CRISPR-GFET biosensor provided by this invention utilizes PDMS / LIG as electrodes and graphene as the semiconductor layer, fabricating a van der Waals contact GFET through a simple one-step bonding method. Gold nanoparticles are used to modify the GFET, and hairpin DNA is used as a reporter, enhancing the electrical signal of the CRISPR-GFET biosensor. On one hand, the simple GFET construction method simplifies the construction steps of the CRISPR-GFET biosensor and reduces detection costs. On the other hand, using a hairpin reporter to construct the biosensor, based on the CRISPR-Cas10 complex-mediated cyclic shearing mechanism and the high charge density of the hairpin reporter, achieves more sensitive detection compared to CRISPR biosensors using straight-chain reporters. Furthermore, integrating a CRISPR system mixing well on the CRISPR-GFET biosensor achieves a high degree of integration in the detection process.

[0061] 5. The detection system based on CRISPR-Cas10 complex and graphene field-effect transistor provided by this invention can complete the detection within 45 minutes of incubation, realizing the universal detection of RNA / miRNA. Attached Figure Description

[0062] Figure 1 This invention describes the sensing mechanism of the detection system based on the CRISPR-Cas10 composite and graphene field-effect transistor.

[0063] Figure 2 This is a schematic diagram of the detection system based on the CRISPR-Cas10 composite and graphene field-effect transistor of the present invention.

[0064] Figure 3 This is a schematic diagram of the detection process of the detection system based on CRISPR-Cas10 composite and graphene field-effect transistor of the present invention.

[0065] Figure 4 This is a curve showing the transfer characteristics of the target RNA detected by the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor of this invention.

[0066] Figure 5 This represents the optimal reaction time for the detection system based on the CRISPR-Cas10 composite and graphene field-effect transistor of this invention.

[0067] Figure 6 The optimal reaction concentration of the CRISPR-Cas10 complex in the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor of this invention is determined.

[0068] Figure 7This invention relates to the detection system for RNA based on the CRISPR-Cas10 complex and graphene field-effect transistor.

[0069] Figure 8 The present invention provides the detection system for RNA based on the CRISPR-Cas10 complex and graphene field-effect transistor.

[0070] Figure 9 The detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor of this invention is used to detect the transfer characteristics of miRNA-155.

[0071] Figure 10 This invention relates to the electrical response of the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor to the concentration of miRNA-155 in serum samples from healthy individuals and breast cancer patients.

[0072] Figure 11 The sensitivity of the re-lDNA CRISPR-GFET biosensor obtained in Comparative Example 1 is shown. Detailed Implementation

[0073] The present invention will be further described below with reference to the embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0074] Example 1

[0075] like Figure 2 As shown, a detection system based on a CRISPR-Cas10 complex and a graphene field-effect transistor includes a CRISPR-Cas10 complex and a CRISPR-GFET biosensor.

[0076] The CRISPR-GFET biosensor includes a sensing substrate, on which a graphene transistor sensing chip is disposed, and on which a microfluidic chip is disposed; the microfluidic chip is provided with a CRISPR system mixing hole and a detection reaction hole connected by a flow channel, and a source electrode and a drain electrode are respectively disposed on both sides of the detection reaction hole.

[0077] Gold nanomaterials and a hairpin report are fixed inside the detection reaction well;

[0078] A gate electrode, which is a silver / silver chloride reference electrode, is also placed inside the detection reaction well.

[0079] The gold nanomaterials (AuNPs) were prepared according to the following method:

[0080] Prepare a 38.8 mM sodium citrate aqueous solution and a 1 mM HAuCl4 aqueous solution. Heat the HAuCl4 aqueous solution to boiling, and then add the sodium citrate aqueous solution to the HAuCl4 aqueous solution while stirring. React for 10 min while heating to boiling. After the reaction is complete, stir for another 15 min, cool to 25 °C, and filter to obtain gold nanomaterials (AuNPs).

[0081] The hairpin reporter is a TCEP-pretreated thiolized hairpin DNA reporter gene, prepared as follows: First, the hairpin reporter was dissolved in annealing buffer (10mM Tris, pH 7.5-8.0, 50mM NaCl, 1mM EDTA) to prepare a 15μM hairpin DNA reporter gene solution. The solution was heated at 95℃ for 2 min, then slowly cooled to 25℃ to form a secondary structure, yielding a thiol-modified hairpin reporter gene. Finally, 200μl of the 15μM thiol-modified hairpin reporter gene was mixed with 5μl of 1M TCEP solution and reduced at 25℃ for 30 min to obtain the TCEP-pretreated thiolized hairpin DNA reporter gene.

[0082] The sequence of the hairpin DNA reporter gene is as follows:

[0083] 5'-TCAGCGTTTCCTTTACCATTTTTTTAACTTATTTGGGTTTTTTTTTT-3'.

[0084] Example 2

[0085] The preparation method of the CRISPR-Cas10 complex described in Example 1 includes the following steps:

[0086] (1) The RNA-F / R / SR primers were used for overlap extension PCR amplification to generate multiple copies of Ld repeat sequence + spacer sequence, which were recovered from the agarose gel to obtain an artificial mini CRISPR fragment of about 1kb; the pUCE plasmid was linearized with EcoRI and SalI, and then PCR amplification was performed using the linearized pUCE plasmid as a template and pUCE-repeat-F / R as primers. The 5' end sequence of the Ld repeat sequence was added to one end of the pUCE fragment (cut by SalI), while the 3' end sequence of the Ld repeat sequence was added to the other end of the pUCE fragment (cut by EcoRI), resulting in a pUCE fragment containing Ld repeat sequences at both ends.

[0087] The sequence of the RNA-F / R / SR is as follows:

[0088] RNA-F:

[0089] 5'-GCTAACAACTTATCTCCGCTAGAAGGAGACGAGAACGCTAGTTACACTA-3',

[0090] RNA-R:

[0091] 5'-GTTCTCGTCTCCTTCTAGCGGAGATAAGTTGTTAGCACACAATCGAAGC-3';

[0092] RNA-SR:

[0093] 5'-TAGCACACAATCGAAGCGCAGTAAGGATGGCTAGTGTAACTAGCG-3';

[0094] The pUCE-repeat-F / R sequence is as follows:

[0095] pUCE-repeat-F:

[0096] 5'-GCTAGAAGGAGACGAGAACAAGCTTGCGGCCGCACTC-3',

[0097] pUCE-repeat-R:

[0098] 5'-GTTCTCGTCTCCTTCTAGCGGAGATAAGTTGTTAGCTTAATGCTAA-3';

[0099] (2) Artificial mini CRISPR fragments were ligated to pUCE fragments containing Ld repeat sequences at both ends by Gibson assembly to obtain pUCE-S-RNA plasmids for specific expression of crRNA targeting RNA.

[0100] (3) pUCE-S-RNA plasmid, pET30a-Csm2 plasmid and p15AIE-Cas-Csm3 plasmid D34A The plasmid was transformed into Escherichia coli BL21(DE3) to obtain an engineered bacterium expressing the CRISPR-Cas10 complex;

[0101] In this engineered bacterium, the pUCE-S-RNA plasmid produces precursor crRNA, which is then processed by p15AIE-Cas-Csm3. D34A Treatment of Cas6 protein expressed in plasmids produces monomeric crRNA; the monomeric crRNA is then combined with pET30a-Csm2 plasmid and p15AIE-Cas-Csm3 plasmid. D34AThe expressed Csm1-Csm5 subunits integrate into the complete CRISPR-Cas10 complex, which can specifically target the target RNA;

[0102] Among them, pET30a-Csm2 plasmid and p15AIE-Cas-Csm3 D34A All plasmids are existing plasmids, which have been disclosed in Chinese patent document CN112760308A-LdCsm-dCsm3 mutant complex, detection system containing the complex and its application in RNA detection;

[0103] (4) The engineered bacteria expressing the CRISPR-Cas10 complex were activated and cultured for 16 h to obtain a culture; then 10 mL of the culture was transferred to 1 L of TB liquid medium and cultured until the mid-colony stage (OD600 = 0.8) was reached; then IPTG was added to a final concentration of 0.3 mM and fermented at 25 °C and 180 rpm for 16 h to induce the production of the CRISPR-Cas10 complex.

[0104] Next, the fermentation culture was centrifuged at 5000 rpm for 5 minutes to collect the cells, and then resuspended in 50 mL of buffer A; then the CRISPR-Cas10 complex that can specifically recognize RNA was collected on the HisTrap affinity column and eluted with buffer B to obtain the CRISPR-Cas10 complex.

[0105] Example 3

[0106] The fabrication method of the CRISPR-GFET biosensor described in Example 1 includes the following steps:

[0107] 1) Two parallel, highly conductive laser-induced graphene (LIG) films were induced on polyimide (PI) using a laser to obtain a LIG / PI film;

[0108] 2) A mixture of polydimethylsiloxane (PDMS) monomer and its curing agent (the mass ratio of PDMS monomer to curing agent is 5:1) is deposited on a silicon mold and cured at 80°C for 60 min to obtain cured PDMS;

[0109] The prepared PDMS was cut into 30mm × 12mm pieces using a laser engraving machine. Two 1.5mm × 1.5mm culture wells, connected by 0.5mm × 3mm channels, were prepared in the middle to serve as mixing wells and detection reaction wells for the CRISPR system, respectively. The laser power and scanning speed were 30W and 30mm·s, respectively. -1 A PDMS electrode adhesion layer was obtained;

[0110] The PDMS electrode adhesive layer is attached to the LIG / PI film, and then the PI film is peeled off to obtain the LIG / PDMS layer.

[0111] The LIG / PDMS layer is then bonded to the graphene / Si / SiO2 substrate to obtain the GFET chip;

[0112] 3) Gold nanomaterials (AuNPs) were dropped into the detection reaction wells of the GFET chip and incubated for 30 min to deposit them in the detection reaction wells of the GFET chip, thus obtaining an AuNPs-modified GFET chip; then, the AuNPs-modified GFET chip was co-incubated with 5 μM CEP pretreated thiolized hairpin DNA reporter gene (re-hpDNA) for 16 h to obtain a re-hpDNA-modified chip; after washing, the re-hpDNA-modified chip was blocked with 2 mM MCH and 1% BSA for 60 min and 30 min, respectively; finally, a silver / silver chloride reference electrode was placed in the detection reaction well to obtain a CRISPR-GFET biosensor.

[0113] In this embodiment, the electrodes of the CRISPR-GFET biosensor are fabricated by preparing LIG on a PI substrate and then transferring the LIG from the PI substrate using PDMS. The transferred LIG serves as the source and drain electrodes. Subsequent testing is achieved by connecting the electrodes with external copper wires.

[0114] Example 4

[0115] In this embodiment, the sample to be tested is 1 μL of 10 pM target RNA, and its sequence information is as follows:

[0116] 5'-AGCGCAGUAAGGAUGGCUAGUGUAACUAGCAAGAAU-3'.

[0117] like Figure 3 As shown, a method for detecting RNA using the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor described in Example 1 includes the following steps:

[0118] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0119] b. Add 1 μL of 10 pM target RNA, 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 1 μL of 200 nM CRISPR-Cas10 complex to 7 μL of DEPC-treated water, mix well to obtain the CRISPR reaction system;

[0120] The concentration of the CRISPR-Cas10 complex in the system was 20 nM, and the concentration of the target RNA was 1 pM.

[0121] The CRISPR reaction system was then added to the mixing well of the CRISPR system of the CRISPR-GFET biosensor and mixed for 5 min. The mixed sample was then blown into the detection reaction well and incubated at 37°C for 60 min. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0122] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. The result is as follows: Figure 4 Place

[0123] Depend on Figure 4 It can be seen that after the addition of target RNA, the lowest voltage (V) in the transfer characteristic curve dirac A positive shift in direction and a positive ΔD value indicate the presence of the target RNA. Therefore, the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor of this invention can detect RNA.

[0124] Example 5

[0125] This embodiment tests the optimal reaction time of the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor. The sample used is 1 μL of 10 pM target RNA, and its sequence information is as follows:

[0126] 5'-AGCGCAGUAAGGAUGGCUAGUGUAACUAGCAAGAAU-3'.

[0127] A method for detecting RNA using the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor described in Example 1 includes the following steps:

[0128] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0129] b. Add 5 μL of 10 pM target RNA, 5 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 5 μL of CRISPR-Cas10 complex to 35 μL of DEPC-treated water, mix well, and obtain the CRISPR reaction system.

[0130] The concentration of the CRISPR-Cas10 complex in the system was 20 nM, and the concentration of the target RNA was 1 pM.

[0131] The CRISPR reaction system was then divided into five equal volumes and added to the mixing wells of the CRISPR system of the CRISPR-GFET biosensor. The mixtures were mixed for 5 min. The mixed samples were then blown into the detection reaction wells and incubated at 37°C for 15, 30, 45, 60, and 75 min, respectively. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0132] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. Statistically analyze the sensor output signal values ​​for different incubation times. The results are as follows: Figure 5 As shown.

[0133] Depend on Figure 5 It can be seen that as the incubation time is extended from 0 minutes to 75 minutes, V dirac The displacement gradually increased; after 45 minutes, as the incubation time increased, V dirac The rate of displacement increase slows down. Considering both time and sensitivity benefits, an incubation time of 45 minutes is the optimal incubation time for the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor.

[0134] Example 6

[0135] This embodiment tests the optimal reaction concentration of the CRISPR-Cas10 complex in a detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor. The sample used is 1 μL of 10 pM target RNA with the following sequence information: 5'-AGCGCAGUAAGGAUGGCUAGUGUAACUAGCAAGAAU-3'.

[0136] A method for detecting RNA using the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor described in Example 1 includes the following steps:

[0137] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0138] b. Add 1 μL of 10 pM target RNA, 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 1 μL of CRISPR-Cas10 complex at concentrations of 100, 150, 200 and 250 nM to 7 μL of DEPC-treated water, mix well, and obtain CRISPR reaction systems with four different concentrations of CRISPR-Cas10 complex;

[0139] The concentrations of the CRISPR-Cas10 complex in the system were 10, 15, 20, and 25 nM, respectively, and the concentration of the target RNA was 1 pM.

[0140] The CRISPR reaction system was then added to the mixing well of the CRISPR system of the CRISPR-GFET biosensor and mixed for 5 min. The mixed sample was then blown into the detection reaction well and incubated at 37°C for 45 min. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove the re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0141] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. Statistically analyze the sensor output signal values ​​at different CRISPR-Cas10 complex concentrations. The results are as follows: Figure 6 As shown.

[0142] Depend on Figure 6 It can be seen that within the concentration range of 10–25 nM, as the concentration of the CRISPR-Cas10 complex increases, the response signal (ΔV) of the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor also increases. dirac The response first increases and then decreases, reaching its maximum at 20 nM, indicating that the optimal CRISPR-Cas10 complex for the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor is 20 nM.

[0143] Example 7

[0144] This embodiment utilizes a detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor to assess RNA sensitivity. The sample used is the target RNA, and its sequence information is as follows:

[0145] 5'-AGCGCAGUAAGGAUGGCUAGUGUAACUAGCAAGAAU-3'.

[0146] A method for detecting RNA using the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor described in Example 1 includes the following steps:

[0147] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0148] b. Add 1 μL of target RNA of different concentrations (0, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM), 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 1 μL of 200 nM CRISPR-Cas10 complex to 7 μL of DEPC-treated water, mix well, and obtain CRISPR reaction systems with 6 different target DNA concentrations;

[0149] The concentrations of the CRISPR-Cas10 complex in the system were 20 nM, and the concentrations of the target RNA were 0, 100 aM, 1 fM, 10 fM, 100 fM, and 1 pM, respectively.

[0150] The CRISPR reaction system was then added to the mixing well of the CRISPR system of the CRISPR-GFET biosensor and mixed for 5 min. The mixed sample was then blown into the detection reaction well and incubated at 37°C for 45 min. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove the re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0151] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. Statistically analyze the sensor output signal values ​​at different target DNA concentrations, and then plot a standard curve based on the obtained ΔD values. The results are as follows: Figure 7 As shown in the figure, y represents the lowest point voltage (V) in the transfer characteristic curve. dirac ), where C represents the target DNA concentration.

[0152] Depend on Figure 7 It is known that the sensitivity of the detection system based on the CRISPR-Cas10 composite and graphene field-effect transistor of this invention is 10.24 mV / decade.

[0153] Example 8

[0154] This embodiment is based on the detection system of CRISPR-Cas10 complex and graphene field-effect transistor for RNA specificity. The test samples used were four different RNAs, 1 μL 10 pM each, and their specific sequences are shown in Table 1.

[0155] Table 1

[0156]

[0157] A method for detecting RNA using the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor described in Example 1 includes the following steps:

[0158] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0159] b. Add 1 μL of 10 pM of each of the four different RNAs listed in Table 1, 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA), and 1 μL of 200 nM CRISPR-Cas10 complex to 7 μL of DEPC-treated water, mix well, and obtain CRISPR reaction systems for the four different target DNAs; at the same time, use an equal volume of DEPC-treated water as a blank system.

[0160] The concentration of the CRISPR-Cas10 complex in the system was 20 nM, and the concentration of the target RNA was 1 pM.

[0161] Then, the blank system and the CRISPR reaction systems of four different target DNAs were added to the mixing wells of the CRISPR system of the CRISPR-GFET biosensor and mixed for 5 min. The mixed samples were then blown into the detection reaction wells and incubated at 37°C for 45 min. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0162] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. Statistically analyze the sensor output signal values ​​for different target DNAs. The results are as follows: Figure 8 As shown.

[0163] Depend on Figure 8 It can be seen that when interacting with blank, non-target RNA-1, RNA-2, RNA-3, and target RNA, ΔV diracThe values ​​were 5mV, 7mV, 9mV, 9mV, and 59mV, respectively, with the electrical response of the target RNA being six times that of the non-target RNA. This result demonstrates that the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor of this invention has good specificity.

[0164] Example 9

[0165] This embodiment uses a detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor to detect miRNA-155.

[0166] The sequence of miRNA-155 is: 5'-UUAAUGCUAAUCGUGAUAGGGGUU-3'.

[0167] A method for detecting miRNA-155 using the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor described in Example 1 includes the following steps:

[0168] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0169] b. Add 1 μL of 10 pM miRNA-155, 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 1 μL of 200 nM CRISPR-Cas10 complex to 7 μL of DEPC-treated water, mix well, and obtain the CRISPR reaction system;

[0170] The concentration of the CRISPR-Cas10 complex in the system was 20 nM, and the concentration of miRNA-155 was 1 pM.

[0171] The CRISPR reaction system was then added to the mixing well of the CRISPR system of the CRISPR-GFET biosensor and mixed for 5 min. The mixed sample was then blown into the detection reaction well and incubated at 37°C for 45 min. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0172] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. The result is as follows: Figure 9 As shown.

[0173] Depend on Figure 9It can be seen that after the addition of miRNA-155, V dirac The forward movement indicates that the detection system based on the CRISPR-Cas10 composite and graphene field-effect transistor of the present invention can detect miRA-155.

[0174] Example 10

[0175] This embodiment uses a detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor to distinguish between healthy individuals and breast cancer patients by detecting miRNA-155 in serum.

[0176] The sequence of miRNA-155 is: 5'-UUAAUGCUAAUCGUGAUAGGGGUU-3'.

[0177] The samples used for testing were: ① 1 μL of serum from a breast cancer patient; ② 1 μL of serum from a healthy person.

[0178] A method for detecting miRNA-155 using the detection system based on the CRISPR-Cas10 complex and graphene field-effect transistor described in Example 1 includes the following steps:

[0179] a. Measure the transfer characteristic curve of the blank CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0180] b. Add 1 μL of breast cancer patient serum, 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 1 μL of 200 nM CRISPR-Cas10 complex to 7 μL of DEPC-treated water, mix well to obtain CRISPR reaction system-1, and perform 6 replicates (P1-P6);

[0181] 1 μL of healthy human serum, 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 1 μL of 200 nM CRISPR-Cas10 complex were added to 7 μL of DEPC-treated water and mixed thoroughly to obtain CRISPR reaction system-2. Five replicates (H1-H5) were performed.

[0182] Then, CRISPR reaction system-1 and CRISPR reaction system-2 were added to the CRISPR system mixing well of the CRISPR-GFET biosensor and mixed for 5 min. The mixed sample was then blown into the detection reaction well and incubated at 37°C for 45 min. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0183] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. Statistically analyze the sensor output signal values ​​for different samples. The results are as follows: Figure 10 As shown.

[0184] Depend on Figure 10 It is known that, since miRNA-155 is upregulated in breast cancer patients, the detection system of CRISPR-Cas10 complex and graphene field-effect transistor has a significantly higher electrical signal response to breast cancer patients than to healthy individuals, indicating that the detection system of CRISPR-Cas10 complex and graphene field-effect transistor of this invention can distinguish between healthy individuals and breast cancer patients.

[0185] Comparative Example 1

[0186] 1. A re-lDNA CRISPR-GFET biosensor, the structure and preparation method of which are shown in Example 3, the difference being that the reporter gene used is different. In this comparative example, re-lDNA is used instead of re-hpDNA. The sequence of the re-lDNA is as follows: 5'-CACACACACACACA-3'.

[0187] 2. The sensitivity of the re-lDNA CRISPR-GFET biosensor in detecting RNA was determined using the following method:

[0188] a. Measure the transfer characteristic curve of the blank re-lDNA CRISPR-GFET biosensor and obtain the Dirac point value D1;

[0189] b. Add 1 μL of target RNA of different concentrations (0, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM), 1 μL of 10× lysis buffer (100 nM Tris-HCl, 15 mM MgCl2, 100 mM KCl and 1.5 mg / ml BSA) and 1 μL of 200 nM CRISPR-Cas10 complex to 7 μL of DEPC-treated water, mix well, and obtain CRISPR reaction systems with 6 different target DNA concentrations;

[0190] The concentrations of the CRISPR-Cas10 complex in the system were 20 nM, and the concentrations of the target RNA were 0, 100 aM, 1 fM, 10 fM, 100 fM, and 1 pM, respectively.

[0191] The CRISPR reaction system was then added to the mixing well of the CRISPR system of the CRISPR-GFET biosensor and mixed for 5 min. The mixed sample was then blown into the detection reaction well and incubated at 37°C for 45 min. After incubation, the solution chamber was rinsed five times with 500 μL of DEPC-treated water to remove the re-hpDNA fragments generated by AuNP surface cleavage. The transfer characteristic curve of the CRISPR-GFET biosensor was measured to obtain the Dirac point value D2.

[0192] c. Calculate ΔD = D2 - D1 and record the resulting Dirac point voltage difference ΔD as the sensor output signal. Then, plot a standard curve based on the obtained ΔD value. The result is as follows: Figure 11 As shown in the figure, y represents the lowest point voltage (V) in the transfer characteristic curve. dirac ), where C represents the target DNA concentration.

[0193] Depend on Figure 11 It can be seen that the sensitivity of the re-lDNA CRISPR-GFET biosensor is 7.91 mV / decade, which is only 77% of the sensitivity of the CRISPR-GFET biosensor (re-hpDNA) in Example 3, indicating that the CRISPR-GFET biosensor (re-hpDNA) in Example 3 has better sensitivity.

[0194] Finally, it should be noted that the above description is only a preferred embodiment of the present invention for those skilled in the art, and is not intended to limit the present invention. Modifications or equivalent substitutions to the technical solutions of the present invention should fall within the protection scope of the appended claims.

Claims

1. A CRISPR-Cas10 complex and graphene field effect transistor based detection system, characterized in that, The CRISPR-GFET biosensor comprises a sensing substrate, a graphene transistor sensing chip arranged on the sensing substrate, and a microfluidic chip arranged on the graphene transistor sensing chip; the microfluidic chip is provided with a CRISPR system mixing hole and a detection reaction hole connected by a flow channel, and the detection reaction hole is respectively provided with a source electrode and a drain electrode on two sides. The gold nanomaterial and the hairpin reporter are fixed in the detection reaction hole, and a gate electrode is arranged. The size of the CRISPR-GFET biosensor is 30mmx12mm; the size of the CRISPR system mixing hole and the detection reaction hole is 1.5mmx1.5mm; the size of the flow channel is 0.5mmx3mm; and the gate electrode is a silver / silver chloride reference electrode or a metal electrode.

2. The detection system of claim 1, wherein, The hairpin reporter is a TCEP pretreated thiolated hairpin DNA reporter gene, which is prepared by the following method: first, dissolve the hairpin reporter in annealing buffer to prepare a 15μM hairpin DNA reporter gene solution, heat at 95℃ for 2min, and then slowly cool to 25℃ to form a secondary structure to obtain a thiol-modified hairpin reporter; finally, mix 200μl of the 15μM thiol-modified hairpin reporter with 5μl of 1M TCEP solution, and reduce at 25℃ for 30min to obtain the TCEP pretreated thiolated hairpin DNA reporter gene.

3. The detection system of claim 1, wherein, The sequence of the hairpin DNA reporter gene is as follows: 5'-TCAGCGTTCCTTTACCATTTTTTTAACTTATTTGGTTTTTTTTTT-3'. The preparation method of the CRISPR-Cas10 complex comprises the following steps:

4. The detection system of claim 1, wherein, (1) overlapping extension PCR amplification of RNA-F / R / S-R primers to obtain an artificial mini CRISPR fragment; PCR amplification of the linearized pUCE plasmid as a template and pUCE-repeat-F / R as primers to obtain a pUCE fragment containing Ld repeat sequences at both ends; (2) Gibson assembly of the artificial mini CRISPR fragment to the pUCE fragment containing Ld repeat sequences at both ends to obtain a pUCE-S-RNA plasmid; (4) activating the engineering bacteria expressing the CRISPR-Cas10 complex and culturing for 12-16h to obtain a culture; then transferring the culture to TB liquid medium and culturing until OD600=0.6-0.8, adding IPTG and continuing to culture for 16-20h, centrifuging, column passing and eluting to obtain the CRISPR-Cas10 complex. (3) pUCE-S-RNA plasmid, pET30a-Csm2 plasmid and p15AIE-Cas-Csm3 D34A The plasmids are transformed into E. coli BL21 (DE3) to obtain an engineered bacterium expressing CRISPR-Cas10 complex. In step 1), the sequence of the RNA-F / R / S-R is as follows:

5. The detection system of claim 1, wherein, RNA-F: 5'-GCTAACAACTTATCTCCGCTAGAAGGAGACGAGAACGCTAGTTACACTA-3', RNA-R: ​ 5'-GTTCTCGTCTCCTTCTAGCGGAGATAAGTTGTTAGCACACAATCGAAGC-3'; RNA-S-R: 5'-TAGCACACAATCGAAGCGCAGTAAGGATGGCTAGTGTAACTAGCG-3'; The sequence of the pUCE-repeat-F / R is: pUCE-repeat-F: 5'-GCTAGAAGGAGACGAGAACAAGCTTGCGGCCGCACTC-3', pUCE-repeat-R: 5'-GTTCTCGTCTCCTTCTAGCGGAGATAAGTTGTTAGCTTAATGCTAA-3'.

6. The detection system of claim 1, wherein, The preparation method of the CRISPR-GFET biosensor comprises the following steps: 1) using a laser to induce two layers of parallel high-conductivity laser-induced graphene film on a polyimide, obtaining an LIG / PI film; 2) depositing polydimethylsiloxane (PDMS) monomer and curing agent on a silicon mold, curing at 75-85°C for 55-65 min, obtaining a cured PDMS; cutting the cured PDMS by laser engraving, and preparing a CRISPR system mixed hole and a detection reaction hole connected in the middle thereof, obtaining a PDMS electrode adhesion layer; pasting the PDMS electrode adhesion layer on the LIG / PI film, then peeling off the PI film, obtaining an LIG / PDMS layer; then bonding the LIG / PDMS layer to a graphene substrate, obtaining a GFET chip; 3) depositing gold nano materials (AuNPs) in the detection reaction hole of the GFET chip, obtaining an AuNPs modified GFET chip; then co-incubating the AuNPs modified GFET chip with a hairpin reporter (re-hpDNA) for 12-16 h, obtaining a re-hpDNA modified chip; after chemical blocking of the re-hpDNA modified chip, obtaining a CRISPR-GFET biosensor.

7. The detection system of claim 1, wherein, In step 1), the mass ratio of the polydimethylsiloxane and the curing agent is 5:1; In step 2), the laser power of the laser-engraving cutting is 25-35 W, and the scanning speed is 25-30 mm·s -1 ; In step 3), the concentration of the hairpin reporter is 1-10 μM; In step 3), the blocking is: first incubating the re-hpDNA modified chip with 2 mM MCH for 1-1.5 h, and then incubating the re-hpDNA modified chip with 1% BSA for 30-60 min.

8. The application of the CRISPR-Cas10 complex and graphene field effect transistor based detection system in detecting RNA or miRNA according to claim 1; The miRNA is miRNA-155; The sequence of the miRNA-155 is: 5'-UUAAUGCUAAUCGUGAUAGGGGUU-3'.

9. A method for detecting RNA or miRNA using the graphene field effect transistor-based detection system of claim 1 based on the CRISPR-Cas10 complex, characterized in that, Comprising the following steps: a, determining the transfer characteristic curve of the blank CRISPR-GFET biosensor, obtaining the Dirac point value D1; b. adding the sample to be detected, the CRISPR-Cas10 complex and the lysis buffer into DEPC water, mixing uniformly to obtain a CRISPR reaction system; then adding the CRISPR reaction system into the CRISPR system mixing well of the CRISPR-GFET biosensor, mixing for 5-10 min; then blowing the mixed sample into the detection reaction well, incubating at 37°C for 15-75 min, cleaning the detection reaction well, measuring the transfer characteristic curve of the CRISPR-GFET biosensor to obtain the Dirac point value D2; c. calculating AD=D2-D1 and taking the obtained difference value Dirac point voltage difference AD as the sensing output signal; when AD is a positive value, the target RNA exists in the sample to be detected; when AD is a negative value or equal to 0, the target RNA does not exist in the sample to be detected.

10. The method of detecting RNA or miRNA according to claim 9, wherein, In step b, the volume ratio of the sample to be detected, the CRISPR-Cas10 complex, the lysis buffer and DEPC water is (1-2):(1-2):(1-2):(4-7); the final concentration of the CRISPR-Cas10 complex in the CRISPR reaction system is 10-25 nM; the dilution factor of the sample to be detected is 0.1-0.2.

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